A ternary complex of hydroxycinnamoyl-CoA hydratase-lyase (HCHL) with acetyl-CoA and vanillin gives insights into substrate specificity and mechanism

Joseph P. Bennett, Lucille Bertin, Benjamin Moulton, Ian J. S. Fairlamb, A. Marek Brzozowski, Nicholas J. Walton, Gideon Grogan

Research output: Contribution to journalArticlepeer-review

Abstract

HCHL (hydroxycinnamoyl-CoA hydratase-lyase) catalyses the biotransformation of feruloyl-CoA to acetyl-CoA and the important flavour-fragrance compound vanillin (4-hydroxy-3-methoxy-benzaldehyde) and is exploited in whole-cell systems for the bio-conversion of ferulic acid into natural equivalent vanillin. The reaction catalysed by HCHL has been thought to proceed by a two-step process involving first the hydration of the double bond of feruloyl-CoA and then the cleavage of the resultant beta-hydroxy thioester by retro-aldol reaction to yield the products. Kinetic analysis of active-site residues identified using the crystal structure of HCHL revealed that while Glu-143 was essential for activity, Ser-123 played no major role in catalysis. However, mutation of Tyr-239 to Phe greatly increased the K-M for the substrate binding. Structures of WT (wild-type) HCHL and of the S123A mutant, each of which had been co-crystallized with feruloyl-CoA, reveal a subtle helix movement upon ligand binding, the consequence of which is to bring the phenolic hydroxyl of Tyr-239 into close proximity to Tyr-75 from a neighbouring subunit in order to bind the phenolic hydroxyl of the product vanillin, for which electron density was observed. The active-site residues of ligand-bound HCHL display a remarkable three-dimensional overlap with those of a structurally unrelated enzyme, vanillyl alcohol oxidase, that also recognizes p-hydroxylated aromatic substrates related to vanillin. The data both explain the observed substrate specificity of HCHL for p-hydroxylated cinnamate derivatives and illustrate a remarkable convergence of the molecular determinants of ligand recognition between the two otherwise unrelated enzymes.

Original languageEnglish
Pages (from-to)281-289
Number of pages9
JournalBiochemical journal
Volume414
Issue number2
DOIs
Publication statusPublished - 1 Sep 2008

Keywords

  • aldolase
  • crotonase
  • enoyl-CoA hydratase (ECH)
  • hydroxycinnamoyl-CoA hydratase-lyase (HCHL)
  • ligand complex
  • vanillin
  • 1.8 ANGSTROM RESOLUTION
  • FERULIC ACID
  • CROTONASE SUPERFAMILY
  • ALCOHOL OXIDASE
  • COENZYME-A
  • PSEUDOMONAS-FLUORESCENS
  • NATURAL-PRODUCT
  • ENZYME
  • BIOSYNTHESIS
  • METABOLISM

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